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A Colorful World – fluorescent 3D live imaging
陳壁彰助研究員（應用科學研究中心）
Introduction
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Since the 17 Centuries when Anton van Leeuwenhoek improved
microscope and successfully observed single-celled organism, the
revolution of optical microscopy has been continuously promoting the
development of modern cell biology by building up the fundamentals
of microbiology. Optical imaging techniques provide much important
information in understanding life science especially cellular structure
and morphology because “seeing is believing . However, the resolution
of optical imaging is limited by the diffraction limit, which is discovered
by Ernst Abbe, i.e. λ /2(NA) (NA is the numerical aperture of the
objective lens). For the last 100 years, biologists and optical scientists are unable to obtain a clear optical image
of biological entities down to molecular level that are smaller than the diffraction limit (around 200-nm in lateral
resolution). With the discovery of green fluorescent protein, the winners of Nobel Prize in Chemistry 2014, E. Betzig,
W. E. Moerner and S. Hell, innovated super-resolution microscopic techniques. Such techniques enable biologists
to visualize nano-sized fluorophores that are beyond the diffraction limit. These techniques do not physically violate
the Abbe limit of resolution but exploit the photoluminescence properties and labelling specificity of fluorescence
molecules to achieve super-resolution imaging. However, these super-resolution techniques limit most of their
applications to fixed or dead samples due to the high laser power needed or slow speed for the localization
process. Free from the suspected artifact caused by fixation process in sample preparation, live fluorescent
imaging is more informative for biologists to elucidate the physiology of living specimens, especially for three threedimensional (3D) imaging.
Challenges in the 3D Live Imaging
When it comes to live imaging, there are always tradeoffs between spatial resolution, temporal resolution, and
phototoxicity based on the photon bank as shown to right. High imaging speeds are required to capture fast cellular
process in order to avoid motion blurring in imaging. High spatial resolution is needed to resolve the structure
details. High signal-to-noise ratios are crucial to have good quality imaging in order to perform computational
image analysis. Long observation periods are needed to catch the biological process happening in the living
specimens. Last but not least, low level of light dose is essential to reduce photobleaching of fluorescent probes
and minimize phototoxicity to the samples. How to deal with the limited photon budget emitted from fluorophores
to have the aforementioned tradeoffs inside live specimens efficiently is made even more difficult when the goal
is to capture subcellular details. Established imaging technologies, such as widefield and confocal microscopy,
have significant limitations for in vivo imaging of biological structure and function. Since they each illuminate the
entire thickness of the specimen, even though high resolution information is obtained from only a single focal plane.
This results in premature photobleaching and phototoxicity, limiting the duration of the imaging and altering the
physiological state of the specimen. Thus, there is a great demand for technology which can uncover the complex
biological phenomena that require 5D (x, y, z, t, λ) information in either a single- or multicellular context.
Novel Approach for bioimging in light microscopy
Widefield and confocal microscopes have been the main working platforms for biologists over the course of
several decades. Widefield, or epi-fluorescence microscope provides fast imaging speed because of widefield
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detection, but it suffers from the “out-of-focus” light resulting in the reduced contrast and poor axial resolution.
By use of physically small pinhole, confocal fluorescent microscope can reject out-of-focus background to have
optical section capability. In this point-scanning based optical system, a beam of laser light is focused into
the specimen to excite the fluorescent markers of interest. The generated fluorescent signal at the focus of the
objective is collected sequentially by scanning the laser excitation focus across the sample to reconstruct the
entire 3D image. Obviously, it is time consuming for large sample or too slow speed to catch fast dynamics process
occurring in the live observing subject. The major disadvantage of these two techniques is very inefficient use
of laser excitation light and fluorescent probes, as well as potentially high photodamage to the sample. Later on,
the discovery of two-photon excitation microscopy uses a special and pricing laser light source, ultrafast laser, to
generate the fluorescent signal exclusively at the focal volume by nonlinearity. In addition to the background free,
the near-IR wavelength is used to excite the fluorescent molecules so that the deeper penetration depth is allowed.
However, in the conventional implementation as a point-scanning technique, it suffers from the same limitation in
speed and signal-to-noise ratio as confocal fluorescent microscopes. For live imaging scanning, it is true that line
scanning improves upon point scanning and plane scanning, in turn, improves upon line scanning with respect
to the detection objective. To address this issue, a new fluorescent imaging technique should be invented for this
purpose. A notable example is light sheet based microscopy. Unlike conventional fluorescent imaging based on
the epi-illumination configuration, light sheet based microscopy uses a separate excitation lens perpendicular
to the widefield detection lens to confine the illumination to the neighborhood of the focal plane. By combining
intrinsic optical sectioning with widefield detection, light sheet microscopy allows fast imaging speed to record
multimegapixel imaging of selected plane in a single exposure of the camera. Instead of point-scanning in confocal
or two photon microscopes, an entire excitation plane formed by the laser illuminates the sample from the side.
Thus, the photons emitted from fluorescent probes at this selected excitation plane is collected with an objective
lens, orthogonal to the excitation light sheet. Although this idea is derived from very ancient thought, along with
the rapid development of instruments such as computer, camera or objectives, the stunning performance has
been demonstrated only recently. By selective plane illumination, light sheet microscopy substantially improves
acquisition speed and signal-to-noise ratio, while minimal photobleaching and phototoxicity. Especial for 3D live
imaging, some light sheet microscopy has been operated to collect high-resolution images rapidly and minimizes
damage to cells, meaning it can image the three-dimensional activity of molecules, cells, and embryos in fine
detail over longer periods than was previously possible; imaging three-dimensional (3D) dynamics for hundreds of
volumes, often at sub-second intervals, at the diffraction limit and beyond.
Light sheet based microscopy
In light sheet microscopy, the light sheet could be a real one, existing simultaneously across the illumination
plane (such as generated by cylindrical lens), or a virtual one, created by a serial scanning long Gaussian beam
formed by an imaging objective with a low numerical aperture (NA) excitation across the pupil plane. A real light
sheet typically requires much lower power so that it takes less photo damage at the cost of spatial resolution in all
dimensions, whereas a virtual light sheet gives more control over the spatial and temporal resolution. The 100-yearold idea of light sheet illumination, once termed “ultramicroscopy”. In ultramicroscopy, optical sections are
generated by stepping the specimen through the illumination plane created by cylindrical lens. This method has
been proved to image the cellular resolution in optical-cleaning tissue such as fixed mouse brains, allowing the
resolution in around micrometer range for small objects (less than 2 mm). A modified version of ultramicroscopy
was presented as selective plane illumination microscopy (SPIM), instead of cylindrical lens, the imaging objective
is used to generate a light sheet with scanned Gaussian beam having thinner thickness around 2~10 um (in
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ultramicroscopy the sheet thickness is ~ tenth of micrometer) to improve axial resolution and toward live imaging
applications. SPIM has been demonstrated in a lot of applications, especially for understanding morphogenesis
with live samples such as fruit fly, zebrafish or mouse with the acquisition rates on the order of 175 million voxels
per second. This performance mainly benefits from the advances in high-speed camera technology, such as the
recent progress in scientific complementary metal oxide semiconductor (sCMOS) technology. This technique has
been proven to be a good tool for imaging embryos noninvasively in 3D over time at single-cell resolution; however,
this commercial Gaussian light sheet microscopy uses too thick light sheet over cellular dimensions to benefit subcellular imaging, which results in the drawbacks of substantial out-of-focus excitation and poor axial resolution. For
example, when imaging a 50-μm-diameter cultured cell, an optimized Gaussian light sheet diverges to a full-width
at half-maximum (FWHM) thickness of ~ 3 μm. Because this is threefold greater than the depth of focus of a highnumerical-aperture (NA) detection objective, substantial out-of-focus excitation remains, and hence the benefits
of background reduction and photobleaching mitigation that are the hallmarks of plane illumination are not fully
appreciated. In response, a ultrathin “non-diffracting” Bessel beam plane illumination was introduced to light
sheet microscopy to have less background, less photobleaching, less photodamage and better axial resolution. A
virtual light sheet plane of submicrometer thickness is created by sweeping such a beam, which is well suited to
non-killing high-speed 4D live cell imaging.
Next, an improved version of Bessel light sheet, lattice light sheet
microscopy is invented for the better spatiotemporal resolution, especially for
temporal resolution, to watch the dynamics happening inside the cell. Ultrathin
light sheets from two-dimensional optical lattices that allowed us to image
3D dynamics for hundreds of volumes, often at subsecond intervals, at the
diffraction limit and beyond. The approach begins with a 2D optical lattice.
Optical lattices are periodic interference patterns in two or three dimensions
created by the coherent superposition of a finite number of plane waves
travelling in certain well-defined directions. Like an ideal Bessel beam, an
ideal 2D lattice is non-diffracting in the sense that it propagates indefinitely in a
direction y without changing its cross-sectional profile, which extends infinitely
in x and z. In either case, this is accomplished by confining the illumination at
the rear pupil plane of the excitation objective to points on an infinitesimally thin
ring. In practice, it is necessary to replace this ring with an annulus of finite
thickness, and to control the thickness of the annulus to confine the pattern in
z, thereby producing a sheet rather than a block of light. However, this also
reduces the extent in y over which the pattern is uniform, so there is a tradeoff
between the thinness of the light sheet and its effective field of view in y.
To create these lattices, a fast switching spatial light modulator (SLM) will
be used, conjugated to the sample plane and placed before the annular mask. The xz cross-sectional electric
field amplitude of the desired theoretical lattice light sheet as a binary phase pattern on the SLM will be displayed.
Incident laser light is then diffracted by the SLM, filtered by the mask, and focused by the excitation objective to
produce a light sheet.
To apply such light sheets to in vivo imaging, in Fig. A, the lattice pattern will be conjugated to the rear pupil
plane (yellow) of excitation objective (left) and an orthogonal detection objective (right) will be suspended from
above with their ends dipped in a shallow media-filled and temperature controlled bath. At an angle (Fig. B) in
the yz plane such that their light cones of excitation (yellow) and detection (red) lie on one side of a horizontally
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mounted cover slip upon which the specimen rests. The lattice light sheet created in the xy plane at their common
foci intersects the specimen obliquely (Fig. C). As the specimen is moved through the light sheet, the fluorescence
thereby generated is recorded as series of 2D images on a camera. These are then assembled into a 3D image,
and the process is repeated to build a 4D data set of cellular dynamics.
Applications of lattice light sheet microscopy
T cell motility and T cell/dendritic cell interactions (shown to the
right) illustrates with high temporal resolution how a T cell (red) partially
envelopes a target dendritic cell (blue) in a matter of seconds. A 2x zoom
of the conjugated plane of the T cell is shown below, illustrating the opening
that forms in 80 seconds. The rate of actin flow in 4D is reported therein,
something that could not be ascertained with spinning disk microscopes.
At a larger scale, the unprecedented resolution in a live vertebrate embryo
how stem cells migrate and differentiate into neurons to form a complex
network in the olfactory system (shown to the right) in longer period of time.
The technique can cover for different length scales at space and time, which
make it more attractive for the biologist.
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